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Capillary Electrophoresis

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Title: Capillary Electrophoresis


1
Capillary Electrokinetic Separations
Lecture Date April 26th, 2007
2
Capillary Electrokinetic Separations
  • Outline
  • Brief review of theory
  • Capillary zone electrophoresis (CZE)
  • Capillary gel electrophoresis (CGE)
  • Capillary electrochromatography (CEC)
  • Capillary isoelectric focusing (CIEF)
  • Capillary isotachophoresis (CITP)
  • Micellar electrokinetic capillary chromatography
    (MEKC)
  • Reading (Skoog et al.)
  • Chapter 30, Capillary Electrophoresis and
    Electrochromatography
  • Reading (Cazes et al.)
  • Chapter 25, Capillary Electrophoresis

3
What is Capillary Electrophoresis?
Electrophoresis The differential movement or
migration of ions by attraction or repulsion in
an electric field
Basic Design of Instrumentation
The simplest electrophoretic separations are
based on ion charge / size
4
Types of Molecules that can be Separated by
Capillary Electrophoresis
Proteins Peptides Amino acids Nucleic acids
(RNA and DNA) - also analyzed by slab gel
electrophoresis Inorganic ions Organic bases
Organic acids Whole cells
5
The Basis of Electrophoretic Separations
Migration Velocity Where v migration
velocity of charged particle in the potential
field (cm sec -1) ?ep electrophoretic mobility
(cm2 V-1 sec-1) E field strength (V cm -1) V
applied voltage (V) L length of capillary
(cm) Electrophoretic mobility Where q
charge on ion ? viscosity r ion radius
6
Inside the Capillary The Zeta Potential
  • The inside wall of the capillary is covered by
    silanol groups (SiOH) that are deprotonated
    (SiO-) at pH gt 2
  • SiO- attracts cations to the inside wall of the
    capillary
  • The distribution of charge at the surface is
    described by the Stern double-layer model and
    results in the zeta potential

Top figure R. N. Zare (Stanford University),
bottom figure Royal Society of Chemistry
Note diffuse layer rich in charges but still
mobile
7
Electroosmosis
  • It would seem that CE separations would start in
    the middle and separate ions in two linear
    directions
  • Another effect called electroosmosis makes CE
    like batch chromatography
  • Excess cations in the diffuse Stern double-layer
    flow towards the cathode, exceeding the opposite
    flow towards the anode
  • Net flow occurs as solvated cations drag along
    the solution

Top figure R. N. Zare (Stanford University),
bottom figure Royal Society of Chemistry
Silanols fully ionized above pH 9
8
Electroosmotic Flow (EOF)
  • Net flow becomes is large at higher pH
  • A 50 mM pH 8 buffer flows through a 50-cm
    capillary at 5 cm/min with 25 kV applied
    potential (see pg. 781 of Skoog et al.)
  • Key factors that affect electroosmotic mobility
    dielectric constant and viscosity of buffer
    (controls double-layer compression)
  • EOF can be quenched by protection of silanols or
    low pH
  • Electroosmotic mobility

Where v electroosomotic mobility ?o
dielectric constant of a vacuum ? dielectric
constant of the buffer ? Zeta potential ?
viscosity E electric field
9
Electroosmotic Flow Profile
- driving force (charge along capillary wall) -
no pressure drop is encountered - flow velocity
is uniform across the capillary
Electroosmotic flow profile
Frictional forces at the column walls -
cause a pressure drop across the column
Hydrodynamic flow profile
  • Result electroosmotic flow does not contribute
    significantly to band broadening like
    pressure-driven flow in LC and related techniques

10
Example Calculation of EOF at Two pH Values
  • A certain solution in a capillary has a
    electroosmotic mobility of 1.3 x 10-8 m2/Vs at pH
    2 and 8.1 x 10-8 m2/Vs at pH 12. How long will
    it take a neutral solute to travel 52 cm from the
    injector to the detector with 27 kV applied
    across the 62 cm long tube?

At pH 2
At pH 12
11
Controlling Electroosmotic Flow (EOF)
  • Want to control EOF velocity

12
Electrophoresis and Electroosmosis
  • Combining the two effects for migration velocity
    of an ion (also applies to neutrals, but with ?ep
    0)
  • At pH gt 2, cations flow to cathode because of
    positive contributions from both ?ep and ?eo
  • At pH gt 2, anions flow to anode because of a
    negative contribution from ?ep, but can be pulled
    the other way by a positive contribution from ?eo
    (if EOF is strong enough)
  • At pH gt 2, neutrals flow to the cathode because
    of ?eo only
  • Note neutrals all come out together in basic
    CE-only separations

13
Electrophoresis and Electroosmosis
  • A pictorial representation of the combined effect
    in a capillary, when EO is faster than EP (the
    common case)

Figure from R. N. Zare, Stanford
14
The Electropherogram
  • Detectors are placed at the cathode since under
    common conditions, all species are driven in this
    direction by EOF
  • Detectors similar to those used in LC, typically
    UV absorption, fluorescence, and MS
  • Sensitive detectors are needed for small
    concentrations in CE
  • The general layout of an electropherogram

Figure from Royal Society of Chemistry
15
CE Theory
The unprecedented resolution of CE is a
consequence of the its extremely high
efficiency Van Deemter Equation relates the
plate height H to the velocity of the carrier gas
or liquid
Where A, B, C are constants, and a lower value of
H corresponds to a higher separation efficiency
16
CE Theory
  • In CE, a very narrow open-tubular capillary is
    used
  • No A term (multipath) because tube is open
  • No C term (mass transfer) because there is no
    stationary phase
  • Only the B term (longitudinal diffusion) remains
  • Cross-section of a capillary

Figure from R. N. Zare, Stanford
17
Number of theoretical plates N in CZE
N L/H H B/v 2D/v v ? E
?V/L Therefore, N L/2D/(?V/L) ?V/2D The
resolution is INDEPENDENT of the length of the
column! Moreover, for V 3 000 V/cm x 100 cm 3
x 104 V D 3 x 10-9 m2/s , and ? 2 x 10-8
m2/Vs, we find that N 100, 000 theoretical
plates.
18
Sample Injection in CE
Hydrodynamic injection uses a pressure difference
between the two ends of the capillary Vc
?P?d4 t 128?Lt Vc, calculated
volume of injection P, pressure difference d,
diameter of the column t, injection time ?,
viscosity Electrokinetic injection uses a
voltage difference between the two ends of the
capillary Qi Vapp( kb/ka)t?r2Ci Q, moles
of analyte vapp, velocity t, injection
time kb/ka ratio of conductivities (separation
buffer and sample) r , capillary radius Ci molar
concentration of analyte
19
Joule Heating
  • Joule heating is a consequence of the resistance
    of the solution to the flow of current
  • if heat is not sufficiently dissipated from the
    system the resulting temperature and density
    gradients can reduce separation efficiency
  • Heat dissipation is key to CE operation
  • Power per unit capillary P/L ? r2
  • For smaller capillaries heat is dissipated due to
    the large surface area to volume ratio
  • capillary internal surface area 2? r L
  • capillary internal volume ? r2 L
  • End result high potentials can be applied for
    extremely fast separations (30kV)

20
Capillary Electrophoresis Applications
  • Applications (within analytical chemistry) are
    broad
  • For example, CE has been heavily studied within
    the pharmaceutical industry as an alternative to
    LC in various situations
  • We will look at just one example detecting
    bacterial/microbial contamination quickly using
    CE
  • Current methods require several days. Direct
    innoculation (USP) requires a sample to be placed
    in a bacterial growth medium for several days,
    during which it is checked under a microscope for
    growth or by turbidity measurements
  • False positives are common (simply by exposure to
    air)
  • Techniques like ELISA, PCR, hybridization are
    specific to certain microorganisms

21
Detection of Bacterial Contamination with CE
  • Method
  • A dilute cationic surfactant buffer is used to
    sweep microorganisms out of the sample zone and a
    small plug of blocking agent negates the cells
    mobility and induces aggregation
  • Method detects whole bacterial cellls

Lantz, A. W. Bao, Y. Armstrong, D. W.,
Single-Cell Detection Test of Microbial
Contamination Using Capillary Electrophoresis,
Anal. Chem. 2007, ASAP Article. Rodriguez, M. A.
Lantz, A. W. Armstrong, D. W., Capillary
Electrophoretic Method for the Detection of
Bacterial Contamination, Anal. Chem. 2006, 78,
4759-4767.
22
Detection of Bacterial Contamination with CE
  • Single-cell detection of a variety of bacteria
  • Why is CE a good analytical approach to this
    problem?
  • Fast analysis times (lt10 min)
  • Readily miniaturized

Lantz, A. W. Bao, Y. Armstrong, D. W.,
Single-Cell Detection Test of Microbial
Contamination Using Capillary Electrophoresis,
Anal. Chem. 2007, ASAP Article. Rodriguez, M. A.
Lantz, A. W. Armstrong, D. W., Capillary
Electrophoretic Method for the Detection of
Bacterial Contamination, Anal. Chem. 2006, 78,
4759-4767.
23
Capillary Electrophoresis Summary
  • CE is based on the principles of electrophoresis
  • The speed of movement or migration of solutes in
    CE is determined by their charge and size. Small
    highly charged solutes will migrate more quickly
    then large less charged solutes.
  • Bulk movement of solutes is caused by EOF
  • The speed of EOF can be adjusted by changing the
    buffer pH
  • The flow profile of EOF is flat, yielding high
    separation efficiencies

24
Advantages and Disadvantages of CE
Advantages Offers new selectivity, an
alternative to HPLC Easy and predictable
selectivity High separation efficiency (105 to
106 theoretical plates) Small sample sizes
(1-10 ul) Fast separations (1 to 45 min) Can
be automated Quantitation (linear) Easily
coupled to MS Different modes (to be
discussed) Disadvantages Cannot do preparative
scale separations Low concentrations and large
volumes difficult Sticky compounds Species
that are difficult to dissolve Reproducibility
problems
25
Common Modes of CE in Analytical Chemistry
Capillary Zone electrophoresis (CZE) Capillary
gel electrophoresis (CGE) Capillary
electrochromatography (CEC) Capillary
isoelectric focusing (CIEF) Capillary
isotachophoresis (CITP) Micellar electrokinetic
capillary chromatography (MEKC)
26
Capillary Zone Electrophoresis (CZE)
Capillary Zone Electrophoresis (CZE), also known
as free-solution CE (FSCE), is the simplest form
of CE (what weve been talking about). The
separation mechanism is based on differences in
the charge and ionic radius of the analytes.
Fundamental to CZE are homogeneity of the
buffer solution and constant field strength
throughout the length of the capillary. The
separation relies principally on the pH
controlled dissociation of acidic groups on the
solute or the protonation of basic functions on
the solute.
Figure from delfin.klte.hu/agaspar/ce-research.ht
ml
27
Capillary Gel Electrophoresis (CGE)
Capillary Gel Electrophoresis (CGE) is the
adaptation of traditional gel electrophoresis
into the capillary using polymers in solution to
create a molecular sieve also known as
replaceable physical gel. This allows analytes
having similar charge-to-mass ratios to also be
resolved by size. This technique is commonly
employed in SDS-Gel molecular weight analysis of
proteins and in applications of DNA sequencing
and genotyping.
28
Capillary Isoelectric Focusing (CIEF)
Capillary Isoelectric Focusing (CIEF) allows
amphoteric molecules, such as proteins, to be
separated by electrophoresis in a pH gradient
generated between the cathode and anode. A
solute will migrate to a point where its net
charge is zero. At the solutes isoelectric
point (pI), migration stops and the sample is
focused into a tight zone. In CIEF, once a
solute has focused at its pI, the zone is
mobilized past the detector by either pressure or
chemical means. This technique is commonly
employed in protein characterization as a
mechanism to determine a protein's isoelectric
point.
29
Capillary Isotachophoresis (CITP)
Capillary Isotachophoresis (CITP) is a focusing
technique based on the migration of the sample
components between leading and terminating
electrolytes. Solutes having mobilities
intermediate to those of the leading and
terminating electrolytes stack into sharp,
focused zones. Although it is used as a mode of
separation, transient ITP has been used primarily
as a sample concentration technique.
30
Capillary Electrochromatography (CEC)
  • Capillary Electrochromatography (CEC) is a hybrid
    separation method
  • CEC couples the high separation efficiency of CZE
    with the selectivity of HPLC
  • Uses an electric field rather than hydraulic
    pressure to propel the mobile phase through a
    packed bed
  • Because there is minimal backpressure, it is
    possible to use small-diameter packings and
    achieve very high efficiencies
  • Its most useful application appears to be in the
    form of on-line analyte concentration that can be
    used to concentrate a given sample prior to
    separation by CZE

31
Capillary Electrochromatography (CEC)
CEC combines the strengths of two powerful
analytical techniques - CE and micro-HPLC.
32
Capillary Electrochromatography (CEC)
R. Dadoo, C.H. Yan, R. N. Zare, D. S. Anex, D. J.
Rakestraw,and G. A. Hux, LC-GC International
164-174 (1997).
33
An Example of CEC
  • Consider a CEC test mixture containing
  • The neutral marker thiourea for indication of the
    electroosmotic flow
  • Two compounds with very different polarities (2
    and 5)
  • Two closely related components (3 and 4) to
    test resolving power

34
An Example of CEC
This separation is carried out on an ODS
stationary phase at pH 8
35
An Example of CEC
The separation carried out on an ODS stationary
phase at pH 2.3
36
Conclusions from the CEC Example
Because the packed length and overall length of
these two capillaries are identical, it is
possible to make a direct comparison of the
performance because the field strength and column
bed length are the same. The EOF has decreased
dramatically between pH 8 and pH 2.3 with the
resulting analysis time increasing from
approximately 5 min to over 20 min at the lower
pH.
37
Electrokinetic Capillary Chromatography
Electrokinetic Chromatography (EKC) a family of
electrophoresis techniques named after
electrokinetic phenomena, which include
electroosmosis, electrophoresis and
chromatography. A key example of this is seen
with cyclodextrin-mediated EKC. Here the
differential interaction of enantiomers with the
cyclodextrins allows for the separation of chiral
compounds. This approach to enantiomer analysis
has made a significant impact on the
pharmaceutical industry's approach to assessing
drugs containing enantiomers.
38
Micellar Electrokinetic Capillary Chromatography
Micellar Electrokinetic Capillary Chromatography
(MECC OR MEKC) is a mode of electrokinetic
chromatography in which surfactants are added to
the buffer solution at concentrations that form
micelles. The separation principle of MEKC is
based on a differential partition between the
micelle and the solvent (a pseudo-stationary
phase). This principle can be employed with
charged or neutral solutes and may involve
stationary or mobile micelles. MEKC has great
utility in separating mixtures that contain both
ionic and neutral species, and has become
valuable in the separation of very hydrophobic
pharmaceuticals from their very polar
metabolites.
Analytes travel in here
Sodium dodecyl sulfate polar headgroup,
non-polar tails
39
Micellar Electrokinetic Capillary Chromatography
  • The MEKC surfactants are surface active agents
    such as soap or synthetic detergents with polar
    and non-polar regions.
  • At low concentration, the surfactants are evenly
    distributed
  • At high concentration the surfactants form
    micelles. The most hydrophobic molecules will
    stay in the hydrophobic region on the surfactant
    micelle.
  • Less hydrophobic molecules will partition less
    strongly into the micelle.
  • Small polar molecules in the electrolyte move
    faster than molecules associated with the
    surfatant micelles.
  • The voltage causes the negatively charged
    micelles to flow slower than the bulk flow
    (endoosmotic flow).

40
Method Development in CE
  • Basic guidance, from the Agilent CE system
    documentation

41
New Technology Electrokinetic Pumping
  • Voltage controlled, pulseless
  • No moving parts or seals
  • Inherently microscale
  • High pressure generation
  • Rapid pressure response
  • Inexpensive
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